Hay fever, more particularly ragweed sensitivity, characterized classically by itching of the mucosa of the nose, mouth, pharynx and eyes, lacrimation, sneezing, watery nasal discharge, coughing and sometimes asthmatic wheezing, afflicts a considerable proportion of humans. Contact dermatitis may also be caused by ragweed pollen sensitivity manifested during the fall of the year. Untreated, the disturbance may disappear until the next season, but it may also become progressively worse, year by year and result in seriously discomforting asthma. Treatment initially entails trial of orally active antihistamines, sometimes in combination with sympathomimettes such as phenylpropanolamine or phenylephrine. The symtoms may thus be partially suppressed but the basic physiological difficulty, the histamine release by the antibody-antigen reaction, persists. For short periods of time, antiinflammatory corticoids may be administered. Commonly these treatments are inadequate or unsatisfactory and resort must be had to a more fundamental treatment, namely desensitization. Desensitization of human patients heretofore was accomplished by injections of extract of the offending pollen, in the case of ragweed sensitivity, extract of the pollen of ragweed (Ambrosia artemisiae-folia), to product in the patient an intercepting protective antibody. This deensitization is undertaken with extreme caution. Commencing with a very dilute preparation of ragweed allergen injected subcutaneously, if there is no local reaction within an hour of injection, the next dose is doubled and administered in 3 or 4 days. To be used in the event of an anaphylactic reaction, there must be available for emergency administration, adrenaline, antihistamines and intravenously administrable antiinflammatory corticosteroids during the testing and densitization procedures. Both the time and precautions accompanying the conventional ragweed densitization procedures are inconvenient.
This invention is concerned with the discovery, manufacture and use of a new and specific polypeptide active pollen immunosuppressant fraction, more particularly a ragweed segment that, upon administration to mammals including humans, effects protection against pollen or ragweed sensitivity without the accompanying and dangerous possibility of anaphylactic shock. Pollen allergen preparations of this invention are made from, illustratively, common ragweed, giant ragweed, rye (Groups I, II and III), June grass, orchard grass, sweet vernal grass, red top, timothy, yellow dock, wheat, corn, sagebrush, blue grass, California annual grass, pig weed, Bermuda grass, Russian thistle, mountain cedar, oak, box elder, sycamore, maple, elm, etc. Ragweed constitutes the most prevalent and noxious of these and is the primary example illustrated herein, the procedure being, of course, applicable to other pollen allergens in a like fashion. The instant new polypeptide active pollen immunosuppressant fractions, in contradistinction from prior art allergens, e.g. ragweed, can be administered in combination with adjuvants thus enhancing effectiveness of treatment without likelihood of anaphylaxis.
Unsuccessful previous attempts to achieve separation of protective and allergic inducing activities for the treatment of pollen sensitivities, particularly in ragweed-sensitive individuals have been reported. For example, Ishizaka, K., et al., J. Immunol. 113: 70-77, 1974 prepared four modifications of the active allergenic fraction of ragweed pollen extract, antigen E, obtained by ammonium sulfate gradient solubilization, namely urea denatured antigen E (UD), its alpha and beta polypeptide chains and a reduced carboxymethylated antigen E (RC). Both UD and RC lost antigenic determinants present in the unmodified antigen E and accordingly could not be used to achieve satisfactory antibody production against antigen E in humans. Each of the four preparations failed to combine with human gamma globulin, human Ig G, against the original native antigen, although they did not induce erythema wheal reactions in ragweed sensitive individuals. Since modified antigens do not induce allergenic reactions in the patients but are capable of stimulating T cells, carrier-specific helper cells, Ishizaka speculated that such modified antigens may change the T cell population without side effects. Subsequently, Ishizaka, et al., J. Immunol., 114: 110-115, 1975, reported that the urea-denatured antigen, UD, and the alpha-polypeptide chain isolated from the denatured antigen E importantly possess their own antigenic determinants different from and lacking in the major determinant of the undenatured molecule. While both native and Ishizaka modified antigens stimulated common T cells, mice immunized with the modified antigen formed a small amount of antibody against antigen E, thereby suggesting either contamination of the modified antigen with native protein or the presence of similar denatured protein in the test antigen E. In any event, Ishizaka states, the results collectively indicate that a major population of B cells stimulated by native antigen is different from the majority of B cells stimulated by the modified antigen. Further work on UD or alpha-polypeptide chain isolated from denatured molecules provided additional evidence that these materials undesirably prime T cells specific for native antigen E (Takatsu, et al., J. Immunol., 115: 1469-1476, 1975 and 116: 1257-1264, 1976). However, it must be recognized that the denatured protein and polypeptide chains obtained from the denatured protein themselves introduce foreign material which could aggravate allergic sensitivity by providing yet another foreign sensitizing material, over and above ragweed antigen, to which the body may react.
Investigation of the immunosuppressive properties of bovine serum albumin treated with pepsin to produce a peptic fragment resulted in a product which did not precipitate with antibody bovine serum albumin bud did inhibit the binding of specific antibody to labeled bovine serum albumin, thus indicating the presence of determinants found in the native antigen. This material could be used before immunization with bovine serum albumin in animals to cause significant suppression of allergic response with the suppression attributed to the presence of specific T suppressor cells in the body which are stimulated by the peptic fragment. By the present process of this invention, the original antigenic determinants are maintained as in the native molecule, Muckerheide A., Pesce, A. J., and Michael, J. G., J. Immunol., 119: 1340-1345, 1977. Further data support the concept that several types of determinants on complex protein antigens participate in the regulation of immune response and that separate determinants on the bovine serum albumin were responsible for immunogenic and suppressive properties of the antigen, Dosa, A., Pesce, A. J. Ford, O. J., Muckerheide, A., and Michael, J. G., Immunol., 38: 509-517, 1979. The art failed to show or suggest the preparation of a pollen desensitizing agent free of anaphylactic reactivity, such as could be used therapeutically.